Mammals require molecular oxygen (O2) for essential metabolic processes including oxidative phosphorylation in which O2 serves as electron acceptor during ATP formation. Systemic, local, and intracellular homeostatic responses elicited by hypoxia (the state in which O2 demand exceeds supply) include erythropoiesis by individuals who are anemic or at high altitude (Jelkmann, Physiol. Rev. 72:449-489, 1992), neovascularization in ischemic myocardium (White et al., Circ. Res. 71:1490-1500, 1992), and glycolysis in cells cultured at reduced O2 tension (Wolfe et al., Eur. J Biochem. 135:405-412, 1983). These adaptive responses either increase O2 delivery or activate alternate metabolic pathways that do not require O2. Hypoxia-inducible gene products that participate in these responses include erythropoietin (EPO) (reviewed in Semenza, Hematol. Oncol. Clinics N. Amer. 8:863-884, 1994), vascular endothelial growth factor (VEGF) (Skweiki et al., Nature 359:843-845, 1992; Banai et al, Cardiovasc. Res. glycolytic enzymes (Firth et al, Proc. Natl. Acad. Sci. USA 91:6496-6500, 1994; Semenza et al, J. Biol. Chem. 269:23757-23763, 1994).
The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and thus blood O2-carrying capacity (Jelkmann, 1992, supra; Semenza, 1994, supra). Cis-acting DNA sequences required for transcriptional activation in response to hypoxia were identified in the EPO 3′-flanking region and a trans-acting factor that binds to the enhancer, hypoxia-inducible factor 1 (HIF-1), fulfilled criteria for a physiological regulator of EPO transcription. In particular, inducers of EPO expression (1% O2, cobalt chloride [CoCl2], and desferrioxamine [DFX]) also induced HIF-1 DNA binding activity with similar kinetics. In addition, inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked induction of HIF-1 activity. Furthermore, mutations in the EPO 3′-flanking region that eliminated HIF-1 binding also eliminated enhancer function (Semenza, 1994, supra). These results support a signal transduction pathway requiring ongoing transcription, translation, and protein phosphorylation in the induction of HIF-1 DNA-binding activity and EPO transcription in hypoxic cells (Semenza, 1994, supra).
EPO expression is cell type specific, but induction of HIF-1 activity by 1% O2 CoCl2, or DFX was detected in many mammalian cell lines (Wang & Semenza, Proc. Natl. Acad. Sci. USA 90:4304-4308, 1993). The EPO enhancer directed hypoxia-inducible transcription of reporter genes transfected into non-EPO-producing cells (Wang & Semenza, 1993, supra; Maxwell et al, Proc. Natl. Acad. Sci. USA 90:2423-2427, 1993). RNAs encoding several glycolytic enzymes were induced by 1% O2, CoCl2, or DFX in EPO-producing Hep3B or nonproducing HeLa cells whereas cycloheximide blocked their induction and glycolytic gene sequences containing HIF-1 binding sites mediated hypoxia-inducible transcription in transfection assays (Firth et al., 1994, supra; Semenza et al., 1994, supra). These experiments support the role of HIF-1 in activating homeostatic responses to hypoxia.
Hypoxia inducible factor-1(HIF-1) is a mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia (Wang, G. L., et al., Proc. Natl. Acad. Sci. USA 92:5510-5514, 1995; Wang, G. L., and Semenza, G. L., J. Biol. Chem. 270:1230-1237, 1995; U.S. Pat. No. 5,882,914). HIF-1 is a basic helix loop-helix protein that binds to cis-acting hypoxia-responsive elements of genes induced by hypoxia (Wang, G. L., and Semenza, G. L., Curr. Opin. Hematol. 3:156-162, 1992; Jiang, B. H., et al., J. Biol. Chem. 272:19253-19260, 1997). The genes that are activated by HIF-1 in cells subjected to hypoxia include EPO, vascular endothelial growth hormone (VEGF), heme oxygenase-1, inducible nitric oxide synthase, and glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase 1 (Semenza, G. L., et al., Kid. Int. 51:553-555, 1997). HIF-1 DNA binding activity and HIF-1 protein concentration increase exponentially as cells are subjected to decreasing O2 concentrations (Jiang, B. H., et al, Am J. Physiol. 271:C1172-C1180, 1996).
HIF-1 also activates transcription of the VEGF gene in hypoxic cells (Forsythe et al., 1996; Iyer et al., 1998). When cultured cells are transfected with pCEP4/HIF-1alpha plasmid under conditions that allow expression of HIF-1aipha from a cytomegalovirus promoter and a reporter plasmid containing the hypoxia response element from the VEGF gene, reporter gene expression is increased in cells under non-hypoxic conditions and there is a dramatic superinduction under hypoxic conditions that is dependent upon the presence of an intact HIF-1 binding site (Forsythe et al., 1996). In embryonic stem cells from a knockout mouse, which lack HIF-1alpha expression, there is no expression of VEGF mRNA in response to hypoxia (Iyer et al., 1998).
HIF-1 is a heterodimer of two subunits, HIF-1alpha and HIF-1beta. The HIF-1alpha subunit is unique to HIF-1, whereas HIF-1 beta (also known as aryl hydrocarbon receptor nuclear translocator, ARNT) can dimerize with other proteins. HIF-1 alpha-subunits are stabilized under hypoxic conditions and are important in regulating genes involved in angiogenesis and glucose metabolism.
Structural analysis of HIF-1alpha revealed that dimerization requires two domains, termed HLH and PAS. DNA binding is mediated by a basic domain (Semenza, G. L., et al., Kid. Int. 51:553-555, 1997). Two transactivation domains are contained in HIF-1alpha, located between amino acids 531 and 826. The minimal transactivation domains are at amino acid residues 531-575 and 786-826 (Jiang, B. H., et al., 1997, supra; Semenza, G. L., et al., 1997, supra). Amino acids 1-390 are required for optimal heterodimerization with HIF1beta (ARNT) and DNA binding. In addition, deletion of the carboxy terminus of HIF-1alpha (amino acids 391-826) decreased the ability of HIF-1 to activate transcription. However, HIF-1alpha (1-390) was expressed at high levels in both hypoxic and non-bypoxic cells in contrast to full-length HIF-1 alpha (1-826) which was expressed at much higher levels in hypoxic relative to non-hypoxic cells (Jiang, B.-H., et al., J. Biol. Chem. 271:17771-17778, 1996). Thus, hypoxia has two independent effects on HIF-1alpha activity: (1) hypoxia increases the steady-state levels of HIF-1alpha protein by stabilizing it (i.e. decreasing its degradation); and (2) hypoxia increases the specific transcriptional activity of the protein (i.e. independent of the protein concentration).